Reversed-phase high-performance liquid chromatography of human haemoglobin chains.

A reversed-phase high-performance liquid chromatographic method for the separation of human haemoglobin chains has been devised. Using a LiChrospher 100 CH-8/2 column and a ternary eluent (acetonitrile-methanol-0.155 M NaCl, pH 2.7) improved resolution was achieved between (delta beta) Lepore, beta A, beta S, alpha, G gamma and A gamma chains within a 60-min linear gradient. The A gamma T chain can also be separated by increasing the gradient time and decreasing the flow-rate. Silanophilic interactions play an important role in the retention mechanism, and NaCl addition was necessary in order to suppress adsorption on free silanols. Increasing the methanol concentration to 10% caused a slight increase in chain retention, probably owing to solvation of the stationary phase. The recovery was 82% and the reproducibility of retention times was as good as +/- 1.5%. Quantitation of chains is likely to be possible by peak area measurement. Owing to its sensitivity, the proposed method may be useful in the diagnosis of haemoglobinopathies and in the study of haemoglobin variants.